Redalyc.First Report of Toxocara Cati in the Domestic Land Snail Rumina

Revista Argentina de Microbiología ISSN: 0325-7541 [email protected] Asociación Argentina de Microbiología Argentina

Cardillo, Natalia; Gonzalez Prous, Cintia; Krivokapich, Silvio; Pittaro, Mariana; Ercole, Mariano; Perez, Matías; Pasqualetti, Mariana; Fariña, Fernando; Rosa, Adriana; Gatti, Graciana; Ribicich, Mabel First report of Toxocara cati in the domestic land snail Rumina decollata Revista Argentina de Microbiología, vol. 48, núm. 3, julio-septiembre, 2016, pp. 206-209 Asociación Argentina de Microbiología Buenos Aires, Argentina

Available in: http://www.redalyc.org/articulo.oa?id=213047799006

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Rev Argent Microbiol. 2016;48(3):206---209

R E V I S T A A R G E N T I N A D E MICROBIOLOGÍA

www.elsevier.es/ram

BRIEF REPORTS

First report of Toxocara cati in the domestic land snail

Rumina decollata

a,∗ b b

Natalia Cardillo , Cintia Gonzalez Prous , Silvio Krivokapich ,

a a a a

Mariana Pittaro , Mariano Ercole , Matías Perez , Mariana Pasqualetti ,

a a b a

Fernando Farina˜ , Adriana Rosa , Graciana Gatti , Mabel Ribicich

Cátedra de Parasitología y Enfermedades Parasitarias, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires,

CONICET, Av. Chorroarin 280, C.P. C1427CWO Buenos Aires, Argentina

Instituto Nacional de Enfermedades Infecciosas, ANLIS ‘‘Carlos G Malbrán’’, Argentina

Received 19 November 2015; accepted 15 April 2016

Available online 24 August 2016

KEYWORDS Abstract The prospective role of the land snail Rumina decollata as a potential paratenic host

of Toxocara cati for domestic cats was studied. R. decollata specimens and cats’ feces were

Toxocara cati;

collected from the open spaces of a Buenos Aires city hospital. Cats’ feces were analyzed and

Rumina decollata;

snails were digested to identify T. cati stages, by morphological and molecular analyses. T. cati

Paratenic host

larval eggs were recovered from 23.5% (4/17) of the sampled feces. Twenty percent of snail

pools (5/25) were conﬁrmed to be positive for Toxocara spp. third larval stage (L3) by PCR.

The mean value of total larvae recovered per gram of snail in all positive pools was 5.1, with a

maximum 33 L3/pool. This is the ﬁrst report of T. cati infective larvae in R. decollata domestic

snail as a paratenic host, since the relationship between infection in snails and in cats’ feces

could be demonstrated in a common environment.

open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).

PALABRAS CLAVE

Primer reporte de Toxocara cati en el caracol doméstico Rumina decollata

Toxocara cati;

Rumina decollata; Resumen Se estudió el rol prospectivo de Rumina decollata como potencial hospedador

Hospedador paraténico de Toxocara cati para los gatos domésticos. Se recolectaron caracoles R.

paraténico decollata y heces de gatos de un hospital de la Ciudad Autónoma de Buenos Aires. Se proce-

saron las heces y los caracoles fueron digeridos para identiﬁcar estadios de T. cati por análisis

morfológico y molecular. El 23,5% (4/17) de las muestras de heces resultaron positivas a huevos

larvados de T. cati. El 20% (5/25) de los pooles de caracoles fue positivo a larvas de tercer

estadío (L3) de Toxocara spp. por PCR. El promedio de larvas totales recuperadas por gramo

∗

Corresponding author.

E-mail address: [email protected] (N. Cardillo).

http://dx.doi.org/10.1016/j.ram.2016.04.004

BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Toxocara cati in Rumina decollata snail 207

de caracol en todos los pooles positivos fue de 5.1, con un máximo de 33 L3/pool. Se trata

del primer reporte de R. decollata como hospedador paraténico de T. cati, puesto que ha sido

demostrada la infección en caracoles y gatos en un ambiente común.

art´ıculo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons.org/licenses/by-

nc-nd/4.0/).

Toxocara spp., the common roundworms of dogs and of snail tissue it was used 0.15 g, pepsin, 0.15 ml concen-

cats, produce eggs that are eliminated with their feces to trated HCl and 15 ml tap water), incubated in a magnetic

◦

the environment, which become a source of infection for shaker (1000 g), for one hour, at 37 C. Afterwards, the ﬂuid

paratenic hosts like mammals (including humans), birds and of the digestion was ﬁltered in a 500 ␮ mesh and cen-

invertebrates . Toxocara spp. larvae are able to migrate trifuged at 1500 rpm for three minutes. The supernatant

through the tissues of their hosts, and in man cause human was discharged and total larvae were counted under the

Toxocariasis . In some countries there are cultural dietary light microscope (10×). Larvae were identiﬁed according to

preferences for raw or undercooked meat of paratenic hosts morphological characteristics described by Sprent , like the

and it became a risk factor for acquiring the human disease. anterior end with a mouth dorsally inclined and a spine-like

Most human infections are asymptomatic but the infec- cuticular thickening forms the ventral margin of a shallow

tion may lead in clinical syndromes known as visceral larva buccal capsule .

migrans (VLM), ocular larva migrans (OLM) and common or Polymerase chain reaction (PCR) ampliﬁcation of larval

covert toxocariasis (CT) . DNA was carried out to conﬁrm Toxocara spp. infection.

Rumina decollata (Linnaeus, 1758) is a pulmonate land DNA isolation and extraction was performed using lysis

◦

snail native to southern Europe, northern Africa and western buffer and proteinase K for two hours at 65 C. The PCR

Asia, and it borders the Mediterranean Sea. In America, it mixture (total volume of 25 ␮l) consisted of 2.5 ␮l of 5 ␮l of

was accidentally and intentionally introduced as biological a single larvae preparation, 2.5 ␮l of 10× PCR Buffer, 0.2 ␮l

control of the garden snail Helix aspersa . In Argentina, it of 2.5 mM each deoxynucleotide triphosphate, 2 U Taq poly-

has been recorded in urban areas of Buenos Aires, Mendoza merase and 1 ␮l (6.25 ␮M) of two pair of primers designed in

2 8

and La Pampa provinces . The snail is omnivorous; they also silico by González Prous et al. (5 -ACGTATGCGTGAGCCG-3

prey upon other land snails, eggs, worms and insects and and 5 -GTGTTTTTGGTTTTTGGCG-3 ). The bioinformatic

feed from decaying fresh vegetable and organic matter like analysis was performed using ITS-1 from ribosomal DNA

animal faeces . from T. canis and T. cati obtained from the public database,

This study was aimed at reporting the presence of T. GenBank. It has been designed in-silico primers in order

cati in a stray cat population living in a public institution to amplify a region that could, after sequencing, reveals

of Buenos Aires city and the evidence of infection in R. species of Toxocara. Direct sequencing was performed on

decollata snail, as potential paratenic host of the parasite an automated Sequencer, using forward and reverse primers

in the same environment. and the Big Dye Terminator kit from Applied Biosystems,

The study site was the open spaces of a public Hospital according to the manufacturer’s instructions. Sequences

from the city of Buenos Aires, inhabited by a feline stray were analyzed by alignment with reference sequences

population. The general characteristics of the surrounding from Basic Local Alignment Search Tool (BLAST) algorithm

fences do not allow the entrance of dogs but permit cats’ optimized for highly similar sequences (megablast) at

access. Seventeen samples of cats’ feces were collected the National Center for Biotechnology Information (NCBI,

individually from the environment of the open spaces sur- Bethesda, MD, USA) (www.ncbi.nlm.nih.gov/blast/).

rounding the buildings of this public Institution. They were DNA was ampliﬁed for 35 cycles, each cycle consisted of

◦ ◦ ◦ ◦

stored at 4 C before being processed. R. decollata snails 1 min at 94 C, 1 min at 55 C, and 1 min at 72 C. Previously

were identified according to the descriptions of Dundee , to the first cycle and after the final cycle longer denaturation

◦ ◦

and 75 adult specimens were collected from the same envi- (3 min at 95 C) and extension (10 min at 72 C) steps were

ronment and were kept in plastic bags . applied, respectively. Ampliﬁcation products were visual-

Fifteen grams of each cat sample feces was weighted ized after electrophoresis on 2.5% agarose gel and visualized

and processed by Benbrook’s technique, with modiﬁcation under an UV transiluminator after staining with ethidium

according to Dolcetti , T. cati eggs were identiﬁed under bromide.

optic microscope (10 ), according to Sprent descriptions. Prevalence of T. cati positive feces was calculated. Pro-

R. decollata snails were processed in 25 pools of three adult portion of R. decollata positive pools and mean value of T.

snails each one, since its low weight made it difﬁcult for cati total larvae (L3) per gram and per pool of snail was

individual processing. They were cleaned individually by reported. The estimation of the conﬁdence intervals (CI) was

brushing the foot and shell in order to remove free liv- created at the 95% conﬁdence level. Statistical analysis was

ing nematodes attached or their eggs. Snails were killed by performed using InfoStat software.

immersion in tepid water for 24 h; shells were removed and T. cati larval eggs were recovered in 23.5% (4/17) of

bodies were weighed, minced with scissors and processed the cat sample feces collected. Most of R. decollata snails

by artiﬁcial digestion technique . Each snail macerated pool were found on buried feces (Fig. 1). Third T. cati larval

was placed in an Erlenmeyer with the digestive ﬂuid (per 1 g stages (L3) were recovered from the 20% (5/25) of snails’

208 N. Cardillo et al.

Figure 3 DNA ampliﬁcation of Toxocara spp. isolated

from R. decollata snails by PCR on 2.5% agarose gel,

using the pair of primers 5 -ACGTATGCGTGAGCCG-3 and 5 -

GTGTTTTTGGTTTTTGGCG-3 : lane 1: marker 50 bp; lane 2:

negative control; lane 3---14: ampliﬁcation of DNA from differ-

Figure 1 R. decollata snails feeding on buried feces from the

ent isolated larvae, each lane corresponds to a single larva; lane

open spaces of a public hospital, Autonomous City of Buenos

15: DNA from T. cati larvae isolated from cats feces, positive

Aires, Argentina.

control.

pools (Fig. 2). The mean value of T. cati total larvae per

No. AJ002437), whereas the nucleotide sequence GenBank

gram of snail recovered in total positive pools was 5.1 lar-

Accession No KR337265 showed a slightly lower similarity to

−

vas (CI 95%: 2.5 to 12.7), Mean value of total L3/pool

this Toxocara species (99%, GenBank Accesión No. KJ777179;

was 8.4 (CI 95%: −8.8 to 25.6), and the maximum was

GenBank Accesión No. AB571303; GenBank Accesión No.

33 L3/pool. There have been sequenced two amplicons of

AB110025, GenBank Accesión No. KJ777163, GenBank Acce-

297 bp in length (Fig. 3) from individual larvae that were

sión No. JF837172, GenBank Accesión No. AJ002436; 98%,

deposited into the GenBank database under Accession Nos.

GenBank Accesión No. AJ002437).

KR337264 and KR337265. BLASTN searches against Gen-

In the city of Buenos Aires, there are free cats popu-

Bank reference sequences for T. cati with a query cover

lations living in semi-wild conditions in public institutions

of 100% displayed high degrees of similarities (100---98%). 13

which offer them shelter and protection . The presence

The DNA sequence GenBank Accession No KR337264 in study

of T. cati (23.5%) in cats feces, conﬁrmed that the life

revealed the highest nucleotide identity to T. cati (100%,

cycle of the parasite takes place in the area. Most of snails

GenBank Accesión No. KJ777179; GenBank Accesión No.

were found feeding on buried feces so coprophagus habits

AB571303; GenBank Accesión No. AB110025; 99% , GenBank

of R. decollata were conﬁrm. It seems that feces provide

Accesión No. KJ777163; GenBank Accesión No. JF837172;

food for snails and protective substrate for T. cati eggs by

GenBank Accesión No. AJ002436; 98%, GenBank Accesión

perpetuating the snail and parasite cycle in contaminated

environments. These conditions would represent a risk for

rodents and birds as paratenic hosts and also for cats. Dubin-

sky et al. reported the importance of earthworms and mice

as natural reservoirs of Toxocara spp. larvae in nature, even

more, as sentinels of environmental contamination, espe-

cially in urban areas, representing an important source of

infection for hunter cats.

Sprent recovered T. cati larvae by experimental infec-

tion of earthworms and cockroaches. Mizgajska et al. ,

obtained an 87% of earthworms, collected from urban gar-

dens and courtyards, positive to Toxocara spp. eggs. Umeche

et al. found Toxocara spp. eggs in domestic ﬂies captured

in Nigeria.

Human cases of toxocariasis acquired by eating raw snails

11 1

were reported in Spain and in Italy . In all cases, the snail

species involved probably was H. aspers; since there is a

popular belief in eating raw snails as a good treatment and

prevention of gastric ulcer so it was traditionally medicinally

1 11

Figure 2 T. cati larva 3 obtained from digested snails col- used . Romeu et al. suggested that the likely mechanism

lected from the open spaces of a public hospital, Autonomous of transmission might have been the ingestion of Toxocara

city of Buenos Aires, Argentina. The arrow shows the anterior spp. eggs mechanically attached to snail bodies, eaten raw

end with a mouth dorsally inclined (45 ). without a thorough cleaning.

Toxocara cati in Rumina decollata snail 209

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